The aim of this project has been to develop in vitro systems for the study of the assembly and folding of the MHC class I molecules. Past studies have focused on coupled in vitro transcription/translation systems for examination of the assembly of the three chains of this macromolecule: the class I heavy chain, beta-2 microglobulin, and antigenic peptide. Recent results with in vitro mutants emphasize the value of such a system in evaluating parameters of assembly. Our current approach is to develop more biochemically defined methods, based on genetically engineered, bacterially produced single chain MHC class I proteins and synthetic antigenic peptides. We have demonstrated that biologically active, immunostimulatory MHC class I peptide complexes can be made by in vitro refolding of completely reduced and denatured molecules, suggesting that this is a feasible approach to produce large scale amounts of homogenous complexes for experimental use.